Tissue engineers have taken several approaches to generate replacement tissues and organs in the laboratory. Generally, autologous tissues are seeded onto a scaffold and expanded in culture. This scaffold should be biocompatible in order to avoid inflammatory responses and rejection of the implanted device, and may be biodegradable so that the artificial material is absorbed, leaving only natural tissue.
Scaffold fabrication techniques include an array of polymer processing techniques such as molding, casting, fiber mesh fabrication, and solid freeform fabrication. All of these methods lack the resolution necessary to fashion the finest features of the organ, such as the capillaries that predominate the circulatory supply. Microfabrication technology such as MEMS, including silicon micromachining and polymer replica molding, provides higher resolution for building tissue and organ scaffolds. The resolution of these techniques is in the 10 nm-1 micron range, which is typically in the range of what is necessary to configure the highest fidelity features of an organ.
The microfabrication process is inherently two-dimensional in nature, and therefore a three-dimensional tissue engineered device can be constructed by stacking multiple layers of cell sheets, optionally with microfabricated membranes interspersed between these layers (or within the individual channels). The interspersed membranes contain pores, which govern small molecule transport. The pore size, porosity and permeability of the membrane can be controlled to provide the desired effect for a particular structure or device.
Within the field of tissue engineering, present methods of achieving complex organization of multiple cell types include two broad general categories. One such class of methods entails the use of biochemical factors, chemical gradients, growth factors and other chemical means to arrange a variety of cells on a substrate (FIG. 1). These chemical techniques typically involve the micropatterning of a substrate for cell and tissue engineering with surface chemistries that enhance adhesion, growth, alignment and other behaviors of specific cell types, and to combine these micropatterns to build up clusters of varied cell types to form the microarchitecture of the target organ. One example of this approach is the use of appropriate growth and signaling factors to arrange endothelial cells and hepatocytes, among other cells, into the sinusoids that comprise the liver. Such methods include the use of self-assembled monolayers (alkenethiols), hydrophilic and hydrophobic patterns on surfaces, patterns of electric charges, thermally responsive polymers, polysaccharides, and cell adhesion factors such as laminin, extracellular matrix (ECM), aminosilanes, combinations of adhesive polymers, and cell growth factors.
The second broad class of methods utilizes precision loading of specific cell types into separate microengineered compartments within a tissue-engineered structure (FIG. 2). Such an approach typically utilizes microfluidic loading, either dynamically or statically, of a network of channels or vessels connected to form a cell compartment, and isolated by appropriate means from all other compartments. In sequential fashion, each compartment is loaded with the specific cell type, and communication between compartments controlled by porous or non-porous barriers (e.g., membranes). Properties of the barrier material are governed by the requirements of the specific cells and tissues in adjacent compartments. For instance, in the case of organ-specific cells such as hepatocytes contained in a compartment adjacent to the endothelial cells comprising the vascular supply, the barrier material, or membrane, physically separates the cell types from adjacent compartments during cell seeding, and readily enables the transfer of oxygen, nutrients and waste products between the two compartments.
The principal disadvantage of both of the aforementioned classes of existing techniques for producing complex tissue engineered structures is the challenge presented by the large, three-dimensional nature of the target tissue or organ for replication. These techniques work well and are quite efficacious when applied to laboratory-scale experimentation in which the resulting tissue-engineered product represents a relatively small assembly of perhaps hundreds or thousands of cells arranged within a single layer or a small number of stacked layers. However, replacement tissues and organs represent on the order of billions of cells of several different types, arranged in perhaps hundreds or thousands of layers.
The first class of methods, in which biochemical patterns are generated on engineered substrates, suffers from several drawbacks. First, there is a need to pattern each layer separately, a process not amenable to batch processing techniques such as lithography and molding, but rather to direct write deposition methods, which are difficult to scale up and accelerate. Therefore the chemical patterning process becomes a layer-by-layer challenge, in which a complex array of chemical factors must be sequentially deposited on each layer while precisely aligning to, and without disturbing, prior layers. Next, the method should be robust to three-dimensional assembly processes such as layer bonding and stacking, so that the three-dimensional assembly of the chemically patterned substrates does not disturb or harm the chemistries and patterns themselves. Finally, surface chemistries may interact with one another, through surface diffusion and other phenomena, thereby reducing or negating the efficacy of the approach.
The second class of methods, in which microfluidic culture of individual cell types is undertaken into a series of separate compartments, does not suffer from all of the same drawbacks as the chemical patterning technique. Since the compartment architecture controls the geometry and relative placement of the cell assemblies, the lack of robustness of chemical techniques is not an issue. Further, the three-dimensional assembly methods do not run the risk of harming the micropatterns, since they are robust physical geometries rather than chemical patterns. However, this microfluidic technique quickly becomes very complex when the number of cell types is increased, and places rigorous demands upon the network geometries. This is because the microfluidic compartments must be arranged in three-dimensions without crossing one another, and complex cell subassemblies may not be amenable to replication by interdigitated non-crossing channel networks. Therefore, techniques in which assemblies of cells can be created without regard to the complex nature of the compartment geometries is desired.